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[Arabidopsis] choice of vectors for pull down

Jose Pedro Fonseca via arab-gen%40net.bio.net (by zepedrof from gmail.com)
Tue Nov 18 10:51:46 EST 2008


Hi all,

I want to confirm interaction of three CBL-like proteins in sugar cane with
a CIPK-like protein. I already have the CBL proteins cloned into pet28a
fused with His tag and induction tests with IPTG worked fine. I had problems
inducing the CIPK-like protein fused to a GST tag in pGEX 4t-1.

Now I need to rethink my cloning strategy for CIPK (choose another vector
with a different tag? order new primers and do cloning at pGEx again?).
Based on the above info would anyone be kind to suggest a new vector or
protocol so I could clone CIPK8 for pull down?

I am assuming that for a pull down experiment all i need is one of my
interacting proteins cloned in a vector that will allow me to "trap" it in
an affinity column (in my case His tag) and then elute this with the target
protein fused to a tag so I can do a western later to confirm interaction
right?

 What about adding a FLAG to the CDS of CIPK when I design primers so I can
use an anti -FLAG antibody later? would it be a nice match to go with His
tagged proteins for a pull down?

Thanks,

JP Fonseca


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